The long-term goal of this project is to understand the function of the mammalian olfactory bulb. The map of the input to the bulb that results from odorant stimulation is well known (e.g. Wachowiak and Cohen, 2001) but there is no analogous map of the output of the bulb (carried by the mitral/tufted cells). A comparison of the input and output maps would define the function(s) carried out by the bulb. That information is needed to make sense of the roles of the many different types of bulb interneurons. It is proposed to measure the output map by specifically labeling the mitral/tufted neurons with a Fluorescent Protein (FP) voltage-sensor using transgenic mice expressing cre recombinase in the mitral/tufted neurons; FP voltage sensors will be expressed either by injecting AAV1 virus that carries a floxed version of the sensor. To observe the mitral/tufted cell signal from many glomeruli we would use wide-field or 2-photon fluorescence imaging focused on their glomerular tufts. A second aspect of understanding the function of the olfactory bulb is to determine the roles of its diverse interneuron types (e.g. Parrish-Aungst et al, 2007). We recorded the odor responses of olfactory bulb interneurons and tried to relate these responses to the activated glomeruli. The results were ambiguous because every odorant activated many glomeruli and thus one could not be certain of the relationship between glomeruli and responding neurons. We then used transgenic mice that express channelrhodopsin-2 in only one olfactory receptor neuron type and activated these receptor neurons by pulsed illumination of the olfactory epithelium. This results in input to a single glomerulus which avoids the confound of widespread glomerular activation. We mapped the response to activation of one glomerulus than then found that higher intensity stimulation sometimes led to lateral effects on nearby glomeruli. We plan to study the cellular basis of this lateral inhibition. There has been a dramatic improvement in the signal size of FP voltage-sensors but the constructs with the largest signal have relatively slow response time constants (t=10 and 50 msec) (Jin et al., 2012). Developing a fast FP voltage-sensor with a large signal would benefit the proposed measurements from mitral/tufted cells and would also be useful to the wider community carrying out optical measurements of brain activity. There are now several fast FP voltage-sensors but they have relatively small signal sizes. It is proposed to make a thorough study of the effect of location of one member of a FRET pair on signal size in an attempt to find sensors with large and fast signals. Voltage sensitive dyes have been used extensively in the study of epilepsy and action potential propagation during cardiac arhythmias (e.g. Matiukas et al, 2006; Mironov et al, 2006). Both kinds of measurements would benefit from the cell-type specificity provided by FP voltage sensors.